β-catenin is a multifunctional protein that plays critical roles in cell adhesion as well as signaling (Moon R T et al., Nat Rev Genet. 5(9):691-701, 2004; Nelson W J and Nusse R. Science. 303(5663):1483-7, 2004). It was first identified as a component of a cell adhesion complex that links transmembrane cadherin proteins and cytoskeleton. It is also a central component of the developmentally important Wnt pathway regulating cell growth and differentiation during embryonic development and tumorigenesis (Gregorieff A and Clevers H. Genes Dev. 19(8):877-890, 2005). In the absence of Wnt, most of the β-catenin in epithelial cells is attached to the plasma membrane, where it is associates with E-cadherin in adherens junctions. Cytosolic β-catenin is located in a multiprotein complex consisting of the adenomatous polyposis coli (APC) protein, axin/conductin, and glycogen synthase kinase-3β (GSK-3β). However, mutations of APC or β-catenin are frequently found in various types of cancer cells (Polakis P. Genes Dev. 14(15):1837-1851, 2000). Mutations in one of the ser/thr-phosphorylation sites of β-catenin stabilize it and lead to transcription of target genes, such as cyclin D1 and c-myc, independent of external Wnt signals (Morin P J et al., Science. 275(5307):1787-1790, 1997; Rubinfeld Bet al., Science. 275(5307):1790-1792, 1997; Tetsu 0 and McCormick F. Nature. 398(6726):422-426, 1999; He et al., Science. 281(5382):1509-1512, 1998). TCF proteins bind to the enhancers of these target genes through their HMG-1 (High Mobility Group-1) DNA binding domains and provide the binding site for β-catenin (Behrens J et al., Nature. 382(6592):638-642, 1996; Morin P J et al., Science, 275(5307):1787-1790, 1997).
Since Wnt signaling is critical for tumor development, the interference of β-catenin-mediated signaling has been proposed as a therapeutic strategy, especially in cancers (Lustig B and Behrens J. J Cancer Res Clin Oncol. 129(4):199-221, 2003; Lee et al., Biochem Biophys Res Commun. 327(1):294-299, 2005). Molecules that could modulate this process would be useful for anti-tumor therapy (Tolwinski & Wieschaus, PLoS Biol. 2(4):486-493, 2004; Lipinski et al., Mol. Ther. 10(1):150-61, 2004). A couple of chemical agents have been reported to disrupt the β-catenin/TCF association in cancer cells (Nath et al., Proc Natl Acad Sci USA. 100(22):12584-9, 2003; Lepourcelet et al., Cancer Cell. 5(1):91-102, 2004). However, since β-catenin is a component of several different protein complexes, more specific tools are needed to selectively disrupt the β-catenin interaction with TCF without affecting the interaction with E-cadherin.
T-cell factor-1 (TCF-1) was originally identified as a T-cell specific transcription factor that bound to specific DNA through its high mobility group-1 (HMG-1) DNA binding domain (M van de Wetering et al., EMBO J. 10: 123-32, 1991; M. Oosterwegel et al., J. Exp. Med. 173: 1133-1142, 1991; H. C. Clevers et al., Immunol. Today 14: 592-597, 1993). Even though transgenic and knockout approaches suggested that TCF-1 was likely to be involved in the expansion of T-lymphocytes, exact functions of the TCF-1 protein in T-cell development need to be understood (M. Oosterwegel et al., Development 118: 439-448; S. Verbeek et al., Nature 374: 70-74, 1990; R. M. Okamura et al., Immunity 8: 11-20, 1998).
TCF family proteins bind to DNA in a sequence-specific manner and they seem to act as architectural proteins for the assembly of other transcription factors (J. J. Love et al., Nature 376: 791-795, 1995). Identification of β-catenin as a potent transcriptional co-activator of TCF family proteins led to a greater understanding of their function (H. Clevers and M. van de wertering, Trends Genet. 13: 485-489, 1997; J. Behrens et al., Nature 382: 638-642, 1996). Since it is highly expressed in various cancer cells, it seemed possible that the formation of a transcriptional complex by an oncogenic β-catenin with TCF might be a central event in cancer cell development (V. Korniek et al., Science 275: 1784-1787, 1997; P. J. Morin et al., Science 275: 1787-1790, 1997; B. Rubinfeld et al., Science 275: 1790-1792, 1997).
The TCF/β-catenin protein complex is also a critical regulator of early developmental events such as axis formation in the Xenopus embryo and Wingless signaling in Drosophila (M. Molenaar et al., Cell 86: 391-399, 1996; M. van de Wetering et al., Cell 88: 789-799, 1997; E. Brunner et al., Nature 385: 829-833, 1997). In addition, it was recently shown that the TCF/β-cadenin complex mediating Wnt signaling seems to be an important pathway in immature thymocyte development (V. Ionnidis et al., Nat. Immunol. 2: 691-697, 2001). These findings began to point to the role of TCF family proteins as critical modulators of the expression of genes that control the decision between proliferation and apoptosis (J. Roose and H. Clevers, Biochem. Biophys. Acta 87456: M23-M37: 1999; J. Roose et al., Nature 395: 608-612, 1998). For example, TCF/β-catenin transcribes genes implicated in cancer development, such as cyclin D1 and c-myc (O. Tetsu and F. McCormick, Nature 395: 608-612, 1998; T. C. He et al., Science 281: 1509-1512, 1998).
Aptamers, which are short single-stranded oligonucleotides, form a three-dimensional structure enabling binding to targets owing to their high affinity and specificity. These aptamers not only specifically bind to target proteins but also successfully disrupt their functions, suggesting that they are very useful for understanding the functions of the target proteins.
Reiterated in vitro selection procedures are able to select specific RNA molecules from random RNA library, and nucleic acids selected by this procedure are generally referred to as aptamers (A. D. Ellington and J. W. Szostak, Nature 346: 818-822, 1990; C. Tuerk and L. Gold, Science 249: 505-510, 1990). Because of the large size (1014-1015) of RNA libraries and the ease of generating RNA molecules by in vitro enzymatic reactions, RNA libraries are superior to other biological or synthetic libraries for selecting high affinity aptamers (E. N. Brody and L. Gold, Rev. Mol. Biotechnol. 74: 5-13, 2000).
Interest in potential uses of RNA aptamers as therapeutics has been increased (Nimjee et al., Trends Cardiovasc Med. 15(1):41-45, 2005). High affinity RNA aptamers can be selected by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure (Ellington et al., Nature 346(6287):818-822, 1990; Brody & Gold, J Biotechnol. 74(1):5-13, 2000). RNA aptamers have an advantage over small chemicals as inhibitors because they usually provide extensive binding surface to target proteins. Pathogenic protein-protein interaction might be a great target for RNA aptamers, because high affinity RNA binds to target protein and interferes its binding to other proteins in the complex. Moreover, RNA aptamer can be expressed in the cells as an intramer using RNA expression vector system (Famulok & Mayer, Chembiochem. 6(1):19-26, 2005).
Descriptions on aptamers can be found in following patents. For example, Korean Patent No. 10-2003-0054412 describes a pharmaceutical agent containing a RNA aptamer for the acceleration of coagulation. Korean Patent No. 10-2002-7009983 describes an aptamer containing a reporter gene which is involved in signaling of homologous ligands in the solution. International Patent WO 2003/027319 describes an aptamer containing two or more nucleobase-containing sequences linked by Watson-Crick or homologous binding. However, any of those descriptions describes on the aptamer interacting with TCF binding site of β-catenin or interacting with DNA binding domain of TCF-1.
Therefore, the present inventors selected β-catenin binding RNA aptamers and stably expressed these aptamers as cell-line specific RNA intramers. The present inventors also confirmed that the expressed RNA intramers could inhibit transcription activity of β-catenin and expression of a target gene. Then, the present inventors further completed this invention by confirming that the in vitro selected TCF-1 binding RNA aptamer (RNA aptamer #10) binds to TCF-1 protein to disrupt the binding with cancer-related genes to disrupt the transcription, so that it can be effectively used for the development of an anticancer agent.